Adverse drug reactions associated with anti-retroviral therapy have emerged that pose significant health complications to HIV/AIDS patients. These complications include hepatotoxicity, which has been reported in ~10% of patients who receive ritonavir-containing protease inhibitor regimens. The mechanism of ritonavir-containing protease inhibitor regimen-induced liver injury is currently unknown. The long-term goal of our research is to improve the safety of anti-retroviral therapy. The objective of this application is to understan the mechanism of hepatotoxicity caused by ritonavir-containing protease inhibitor regimens. In a recent series of clinical trials, the hepatotoxicity of ritonavir-containing protease inhibitor regimens was significantly enhanced in subjects who were pretreated with rifampicin, a first-line anti-tuberculosis drug. Rifampicin is also known as an activator of human pregnane X receptor (PXR) that regulates the gene transcription of a large number of proteins including CYP3A. We recapitulated the hepatotoxicity associated with rifampicin pretreatment followed by ritonavir-containing protease inhibitor regimens in double transgenic human PXR and CYP3A4 mice. We found: (1) Human PXR is critical in the hepatotoxicity associated with rifampicin pretreatment followed by ritonavir-containing protease inhibitor regimens, and (2) Ritonavir is the key component that causes the hepatotoxicity of ritonavir-containing protease inhibitor regimens. In addition, CYP3A was determined as the primary enzyme contributing to ritonavir bioactivation. We also observed endoplasmic reticulum (ER) stress, unfolded protein response, and apoptosis in livers of double transgenic human PXR and CYP3A mice pretreated with rifampicin followed by ritonavir. We hypothesize that PXR activation increases ER load and potentiates CYP450-mediated ritonavir bioactivation, which synergizes ER stress and results in hepatotoxicity. To test this hypothesis, the following three specific aims are proposed: (1) Identify the metabolic enzyme(s) mediating the hepatotoxicity associated with rifampicin pretreatment followed by ritonavir; (2) Elucidate the metabolic pathway(s) of ritonavir that are associated with ritonavir-induced hepatotoxicity; and (3) Determine the role of ER stress in the hepatotoxicity of ritonavir-containing protease inhibitor regimens. The results from this project are expected to provide a mechanistic understanding of liver injury associated with ritonavir-containing protease inhibitor regimens, which will be applied toward the development of an evidence-based approach to improve the safety profile of HIV protease inhibitors.